Rift Valley fever Phlebovirus (RVFV), a bunyavirus, belongs to the NIAID Category A list of pathogens and the CDC list of potential bioterrorism agents. RVFV causes a disease that is endemic in sub-Saharan Africa and can emerge in explosive, mosquito-borne epidemics that decimate herds of sheep and cattle, resulting in enormous economic losses. In humans, RVFV infection may cause hemorrhagic fever, encephalitis, and retinal vasculitis. Many different mosquitoes, including several species native to North America, are competent vectors for RVFV transmission. The introduction of RVFV into North America would likely cause panic in the general population, and the effects on livestock could have a devastating economic impact. The lack of availability of licensed vaccines or anti-RVFV reagents for use in humans or domestic animals is of great concern. RVFV carries a tripartite, single-stranded, negative-sense RNA genome, including L RNA encoding L protein, M RNA encoding two major envelope glycoproteins, Gn and Gc, and S RNA encoding N and NSs. NSs is the major virulence factor that efficiently suppresses host antiviral responses. The S RNA uses an ambi-sense strategy for gene expression; NSs is translated from the mRNA that is transcribed from the antigenomic-sense S RNA, whereas N protein is produced from the mRNA that is transcribed from the genomic-sense S RNA. One of the essential steps in virus replication and dissemination is the packaging of viral genome into virus particles, however, the mechanisms of viral RNA packaging in RVFV and other bunyaviruses are largely unknown. Insight into the underlying rules and mechanisms that govern the packaging of viral RNA genome into RVFV particles is valuable for understanding the regulation of virus replication, virus evolution, and the pathogenesis of the virus. This knowledge is also critical for the development of antiviral drugs that can inhibit infectious virus production or for the development of a live- attenuated vaccine strain. We propose that a direct interaction of Gn with the viral RNA segments is the primary factor that influences the packaging efficiencies of viral RNAs into RVFV particles. The present application will clarify the mechanism and biological significance of efficient packaging of antigenomic S RNA into virus particles by characterizing the direct interaction of Gn with antigenomic S RNA, identify the Gn- binding sites in viral RNAs, and examine the importance of antigenomic S RNA packaging for RVFV replication. The data obtained from the proposed studies will clarify the fundamental mechanisms that drive viral RNA packaging in RVFV and other bunyaviruses, with the overall goal of informing the design of novel antivirals and vaccines.